From the Biochemistry Section and.
Structural Biochemistry, Department of Biology/Chemistry, University of Osnabrück, Barbarastrasse 13, 49076 Osnabrück, Germany.
J Biol Chem. 2018 Jan 12;293(2):731-739. doi: 10.1074/jbc.M117.812941. Epub 2017 Nov 28.
The identity of organelles in the endomembrane system of any eukaryotic cell critically depends on the correctly localized Rab GTPase, which binds effectors and thus promotes membrane remodeling or fusion. However, it is still unresolved which factors are required and therefore define the localization of the correct fusion machinery. Using SNARE-decorated proteoliposomes that cannot fuse on their own, we now demonstrate that full fusion activity can be achieved by just four soluble factors: a soluble SNARE (Vam7), a guanine nucleotide exchange factor (GEF, Mon1-Ccz1), a Rab-GDP dissociation inhibitor (GDI) complex (prenylated Ypt7-GDI), and a Rab effector complex (HOPS). Our findings reveal that the GEF Mon1-Ccz1 is necessary and sufficient for stabilizing prenylated Ypt7 on membranes. HOPS binding to Ypt7-GTP then drives SNARE-mediated fusion, which is fully GTP-dependent. We conclude that an entire fusion cascade can be controlled by a GEF.
真核细胞内膜系统中细胞器的身份取决于正确定位的 Rab GTPase，它结合效应因子，从而促进膜重塑或融合。然而，目前仍不清楚需要哪些因素，因此也无法确定正确融合机制的定位。我们使用自身不能融合的 SNARE 装饰的类脂蛋白体，现在证明仅通过四种可溶性因子就可实现完全融合活性：一种可溶性 SNARE（Vam7）、一种鸟嘌呤核苷酸交换因子（GEF，Mon1-Ccz1）、一种 Rab-GDP 解离抑制剂（GDI）复合物（被异戊烯基化的 Ypt7-GDI）和一种 Rab 效应物复合物（HOPS）。我们的研究结果表明，GEF Mon1-Ccz1 对于稳定质膜上的异戊烯基化 Ypt7 是必需且充分的。HOPS 与 Ypt7-GTP 的结合随后驱动 SNARE 介导的融合，这完全依赖 GTP。我们得出结论，整个融合级联反应可以由 GEF 控制。
